1. Field of the Invention
The present invention relates to a process for preparing peptide nucleic acid probes by employing polymeric photoacid generator, more specifically to a process for preparing arrays of oligopeptide nucleic acid probes immobilized on a solid matrix by employing polymeric photoacid generator.
2. Description of the Prior Art
Biochip, a kind of biosensor used for genomic analysis, medical diagnosis and monitoring biological processes and environment, is largely classified into DNA chip which identifies nucleotide sequences and protein chip which recognizes proteins such as pathogens, antibodies, antigens or enzymes.
The structure of DNA identified by Watson and Crick in 1953 has been a great impact on life sciences such as molecular biology, biochemistry, etc. DNA is a biopolymer with four different bases of adenine(A), cytosine(C), guanine(G), thymine(T), sugar(deoxyribose) and phosphate, to build a very stable double helix structure: the phosphate-sugar forms backbone, bases attached to the sugar are paired with complementary bases, such as A to T, and G to C, which are stabilized by hydrogen bond. The specific/complementary hydrogen bond between bases plays a very important role in drug developments such as antisense drug and gene therapy, in particular, for genetic disease, cancer and cardiac diseases.
Recently, in line with the great efforts made to identify approximately 100,000 human genes, needs for the methods for providing enormous genomic information as fast as possible for the diagnosis and prevention of genetic disease are increasing. Despite Sanger""s DNA sequencing method is improved by polymerase chain reaction and automation, it still remains cumbersome and time-consuming, and requires high cost as well as highly trained personnel. Therefore, alternative method to overcome those shortfalls has been constantly deliberated in the art. By virtue of such needs, there has been a considerable progress in the fabrication and utilization techniques of DNA chips for recent several years.
In general, DNA chip is high-density micro-arrays of known oligonucleotide probes ranging from several hundred to several hundred thousands immobilized on a smaller than one square inch solid surface, such as silica, surface-derivatized glass, polypropylene or activated polyacrylamide. If the target DNA fragments are placed on DNA chip, the target DNA fragments hybridize to the probes on the chip according to the sequence complementarity. Therefore, the sequence of the target DNA can be analyzed by the presence and location of hybridized DNA detected by the optical or radiochemical method. Using the DNA chip aided method, DNA analysis system can be miniaturized such that an extremely small amount of sample DNA can be used for diagnosis, several sequences in a target DNA can be analyzed simultaneously, and thus the genetic information can be obtained in a simple, cost effective and fast manner.
The sequence analysis technique using oligonucleotide DNA chip is an innovative method since it is faster and easier to use for sequencing a target gene than the conventional Sanger""s method. The Sanger""s sequencing method, which requires the separation of fluorescence- or radioisotope-labeled DNA fragments by gel electrophoresis, is proven to be less satisfactory in light of time-consuming and technical difficulties. On the contrary, sequencing by hybridization(SBH) using oligonucleotide DNA chip employs a principle that by placing fluorescence- or radioisotope-labeled target gene fragments to a DNA chip with oligonucleotide probes, and then simply by washing the chip with solvent, gene fragments complementary to known oligonucleotide probes are attached by hydrogen bond.
Merryfield""s synthetic method in which chemical compounds are reacted on organic solvent-resistant solid matrix and further organic reactions are carried out on the solid matrix, has been used effectively in synthesizing oligopeptide of biologically important enzymes and oligonucleotide of genes, since it is very simple and efficient in the purification of reaction products (see: Fed. Proc., 24:412, 1962). A new efficient synthetic method combining the said method with combination chemistry has been used effectively for catalyst screening and also been used in drug development as well. In addition, by the combination of the synthetic method on a solid matrix and the photolithography employed in semiconductor industry, various techniques for preparing arrays of oligonucleotide probes have been developed to be used for genetic diagnosis. The said technique comprises selective activation of specific regions of surface-derivatized glass to which the oligonucleotide binds chemically. To activate the specific targeted region, the light beam is applied through a transparent region of photolithographic mask made by a predetermined pattern. By controlling the mask pattern and composition of nucleic acids at each step, a specific nucleic acid can be positioned on a desired location. This technique is an ultra-fine processing technique used in semiconductor devices which makes possible several millions of probes be affixed on a fingernail-size chip.
For example, Fordor et al teaches a new direct photolysis technique, where nucleic acid or amino acid with UV-labile protecting group is attached on a solid surface, the protecting group is eliminated by exposing the selected regions of surface to light using photolithographic mask, which is subsequently reacted with a new nucleic acid or amino acid with a photolabile protecting group, to polymerize nucleic acid or amino acid at specific location(see: U.S. Pat. No. 5,445,934). Since this method allows selective synthesis of oligonucleotide probes with a specific sequence/length at a specific location, it is useful in synthesizing various oligonucleotide probes with a desired sequence and length at a predetermined position. Also, since this method employs ultra-fine process used in semiconductor devices, it is extremely useful for fabricating oligonucleotide probes in high density. Fordor et al also suggested that a sequencing method using the oligonucleotide probes which is much easier and faster than Sanger""s method, is useful for making high density oligonucleotide probes. However, it has revealed a shortcoming that: the removal of photolabile protecting group is proportional to the power of a light source, which plays a detrimental role in the ultra-fine process for making high density chips.
On the other hand, photolithographic process using photoresist(hereinafter referred to as xe2x80x98PRxe2x80x99) which is used for micropattern formation in semiconductor industry has attracted attention as an essential technique to improve the density of DNA chip. Since the size or capacity of a semiconductor chip is depending on the spatial resolution of photolithographic process, photolithographic process has played a leading role in the semiconductor and microelectronics industry. The photolithographic process utilizes the solubility differences of PR between the light exposed region and the unexposed region. The solubility reduction in the exposed region is called as negative system and solubility increase is called as positive system, and the latter is used mostly for the production of semiconductor chips. By using above photolithographic process, more oligonucleotide probes can be arrayed on a limited area of chip.
Up to now, photolithographic process has been applied in the general PR system(see: U.S. Pat. No. 5,658,734) and the photoacid patterned array system(hereinafter referred to as xe2x80x98PPAxe2x80x99, U.S. Pat. No. 5,843,655) as well.
Photolithographic process using PR system(hereinafter referred to as xe2x80x98PR processxe2x80x99) has an advantage of using materials already developed or commercialized for semiconductor industry. According to the system, a pattern is formed by the light exposure and washed out to lead to standard solid-phase nucleic acid synthetic reaction on the surface, finally to link nucleotides. The PR includes diazoquinone/cresol-novolac, highly adhesive to the surface, which is shown to have good pattern characteristics in i-line(365 nm) and used in 16 mega DRAM processing. However, the PR is developed in alkaline solution ([OHxe2x80x94] greater than 0.1M), that causes the cleavage of amide bond protecting the amino group of base. The protecting coating under PR has been suggested to overcome the said problem of development(see: J. Vac. Sci. Tech., B7(6):1734, 1989).
PR process is consisted of three major steps: the first is mainly the PR pattern formation with PR coating, light exposure and developing; the second is the removal of protecting group and unexposed area by acidic solution; and, the third is the attachment of nucleic acid and post-treatment. Photolithographic process using PR system is good enough for obtaining high resolution, but complex processing as described above has been a major drawback.
To simplify the complex process and improve the efficiency, the PPA system has been proposed in the art. Since the PPA system used polymeric matrix mixed with photoacid generator(hereinafter referred to as xe2x80x98PAGxe2x80x99), acids are generated only at the exposed region and removal of protecting group occurs after heat treatment, therefore the two separate steps employed in PR processing can be carried out in one step. However, this PPA system has revealed several problems as well. For example, while PR process is not affected by the concentration or volume of the acid solution since it uses large volume of acid solution to contact and to remove the protecting group, acid generated by the PPA system remains on the polymeric matrix and, to balance the amount of acid, more PAG should be added. However, at above a certain level of PAG, PAG domain forms and scatters light which in turn interferes with micro pattern formation. In other words, excessive amount of PAG which is used for stability and reproducibility of the pattern formation reduces the efficiency of processing. In addition, the generated acid should diffuse from the polymer to the surface(actually closer to the heat source) of the protecting group by heat, but the generated acid, when the glass plate is heated on a hot plate, may diffuse to the opposite direction from the protection group, thereby reducing the efficiency. Furthermore, the mixed film of polymer and PAG for coupling, has to be removed by organic solvent such as acetone or methylethylketone, which makes the progress more expensive.
Another problem confronted in the art DNA chip techniques is the reduction of degrees of the association/dissociation, speed in the course of hybridization of oligonucleotide probes with target genes and point mutations present in a gene. For example, Nielsen et al developed neutrally charged peptide nucleic acid(hereinafter referred to as xe2x80x98PNAxe2x80x99) to replace oligonucleotides which can be synthesized by the modified Marryfield""s peptide synthesis method, based on the findings that the repulsive force between negatively-charged oligonucleotides reduces the annealing strength as well as speed during hybridization of oligonucleotides. PNA greatly improved the association power, and speed during hybridization with oligonucleotide and it is practically applied in detecting single base mutation in a particular gene, which can be realized by the reduced association between non-complementary oligonucleotide(see: Nature, 8:53, 1993). Due to this positive characteristic, the antisense drug using PNA is under investigation. However, despite the benefits, there has been no report of using PNA on high density peptide nucleic acid probe synthesis by photolithographic process.
Therefore, there are strong reasons for developing and exploring alternative method for synthesizing peptide nucleic acid as well as oligonucleotide with various nucleotide sequences on a solid matrix, while overcoming the problems confronted in the prior art DNA chip fabrication using PR and PPA system.
The present inventors made an effort to provide an efficient method for preparing nucleic acid probes with various nucleotide sequences, and developed a highly efficient process for preparing arrays of peptide nucleic acids, which can eliminate the repulsion problem associated with negatively charged oligonucleotide, immobilized on a solid matrix by employing polymeric photoacid generator(hereinafter referred to as xe2x80x98polymeric PAGxe2x80x99) while overcoming the problems of the prior art PPA process employing PAG in polymer.
A primary object of the present invention is to provide a process for preparing arrays of peptide nucleic acid probes with various nucleotide sequences on a solid matrix by employing polymeric photoacid generator.
The other object of the present invention is to provide arrays of peptide nucleic acid probes with various nucleotide sequences prepared by employing polymeric photoacid generator.
The peptide nucleic acid probes with various nucleotide sequences are prepared by derivatizing the surface of a solid matrix with aminoalkyloxysilane in alcohol and attaching a linker with acid-labile protecting group on the solid matrix, coating the solid matrix with polymeric photoacid generator(PAG), exposing the solid matrix thus coated to light to generate acid for eliminating acid-labile protecting group, washing the solid matrix with alkaline solution or organic solvent and removing residual polymeric photoacid generator, and, attaching a monomeric peptide nucleic acid with acid-labile protecting group to the solid matrix, and repeating the previous steps.
Polymeric PAG employed in the invention is a polymer with molecular weight of 500 to 1,000,000, which has sulfonium salt on backbone or side chain, and has also organic photoacid generating group on side chain to generate acid by exposing to light.
The peptide nucleic acid monomers of the invention has N-(2-aminoethyl) glycine backbone to which adenine, cytosine, guanine, or thymine base is linked by amide bond. Peptide nucleic acids are synthesized by amide bond between an amino group of backbone and a carboxyl group of other peptide nucleic acid monomer. Currently, peptide nucleic acid monomers protected by acid-labile t-butyloxycarbonyl protecting group or alkali-labile fluoromethyloxycarbonyl protecting group are commercially available, where exocyclic amino groups of adenine, cytosine and guanine are protected by acid-stable diphenylmethyloxycarbonyl or benzyloxycarbonyl protecting group. The present inventors employed monomers whose amino groups of backbone are protected by acid-labile t-butyloxycarbonyl, and synthesized bases whose exocyclic amino groups are protected by stable p-methoxybenzoyl or isobutanoyl group.
The peptide nucleic acid synthesis method is generally carried out in a similar manner as the oligonucleotide synthesis method conventionally known in the art(see: Acc. Chem. Res., 24:278, 1991). Nielson et al synthesized oligopeptide nucleic acid by using solid-phase phase as follows: First, the amino group of solid support is reacted with the carboxyl group of peptide nucleic acid of specified base(A, C, G or T) whose amino group in backbone is protected by acid- or base-labile functional group to link each other in a form of amide bond. Next, the resultant is treated with acid or base to eliminate amino protecting group to reveal amino group, which is subsequently reacted with the carboxyl group of peptide nucleic acid of specified base whose amino group in backbone is protected by acid- or base labile functional group to link each other in a form of amide bond, and, the said steps are repeated to obtain an oligonucleotide of desired base sequence and number, and finally treated with strong acid to separate the exocyclic amino protecting group from solid support by chemical reaction. This method is desirable in a sense that it assures complete reaction of excessive peptide nucleic acids(5 equivalents) as much as possible and easy purification of peptide nucleic acid on an organic solvent-resistant solid support by filtering the residual monomers and reactants and washing with organic solvent. Based on the solid-phase synthesis, the present inventors prepared biochip with various nucleotide sequences to finally prepare arrays of peptide nucleic acid probes immobilized on a solid matrix by employing polymeric PAG process illustrated above.
The polymeric PAG and peptide nucleic acid monomer which are employed in the probes of preparing peptide nucleic acid probes with various nucleotide sequences were prepared by Preparative Examples 1 and 2, and Preparative Example 3, respectively.